We have been studying clathrin-independent forms of endocytosis (CIE) and have identified a number of endogenous PM proteins that enter cells through this mechanism. We have begun to study these proteins in detail in an attempt to understand how they travel in cells and whether they specifically interact with cellular machinery. To further understand the mechanism or mechanisms of CIE, we have embarked on an siRNA screen using the Dharmacon Trafficking Library that targets 140 human genes. We are looking for hits that affect endocytosis of two CIE cargo proteins: MHCI (major histocompatibility complex Class I) and CD59 (a GPI-lipid anchored protein). We are in the process of validating the hits and will follow up on promising ones. Interestingly, we find that the hits that affect MHCI vs. CD59 endocytosis are different, suggesting that the requirements for internalization of these 2 CIE cargo proteins are somewhat distinct. In another study, we are examining the role of glycosylation on cargo proteins and whether over or under glycosylation will affect rates of endocytosis and the trafficking of those proteins. Here again, the effect of glycosylation of cargo and presence of extracellular lectins can alter the landscape to be permissive or not for certain cargo. Finally we are looking at CIE in different cell types and also in cells that undergo a stimulated form of CIE, macropinocytosis. We previously described alterations in phosphoinositol lipids in HeLa cells expressing active forms of Ras and new work has shown that some cancer cells use macropinocytosis for nutrient uptake. We are now examining the trafficking and sorting of cargo in fibrosarcoma cells undergoing constitutive macropinocytosis and the role that the microtubule cytoskeleton and motors play in this process. In collaborations with Ed Korn's group in NHLBI, we provide evidence that myosin II associates with anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate. Additionally in a collaborative study with Dr. Jia Song at the University of Delaware, we helped establish a role for Arf6 in sea urchin development.